Volume 2 number 10 October 1975

Nucleic Acids Research

Mapping the transcription site of the SV40-specific late 16 S mRNA

E. May*, H. Kopecka** and P. May* *Unite de Biophysique, Institut de Recherches Scientifiques sur le Cancer, BP No. 8, 94 800-Villejuif, France

Received 15 September 1975 ABSTRACT This paper describes the purification of polysomal RNA from

SV40-lytically infected Cvl (monkey) cells and separation of the two distinct classes of SV40-specific mRNA sedimenting at 16 S and 19 S. These classes have been hybridized with the whole SV40 DNA genome as well as with the SV40 Hind fragments. The results have permitted the mapping of SV40specific late 16 S mRNA from approximately 0.945 to 0.175 map units.

INTRODUCTION During the early phase of lytic infection, before the onset of viral DNA replication, viral cytoplasmic RNA is found to sediment in a sucrose gradient as a uniform band corresponding to 19 S (1). The same sedimentation profile is obtained for viral polysomal RNA in SV40-abortively infected mouse kidney cells (2) and in SV40-transformed cells (3). These SV40-specific 19 S RNAs appear to consist mainly of the contiguous transcript of the early region of the viral genome (4, 5, 6) that includes all of Hind fragments A, H, I, B (4-5-7) (48 Z of the viral genome). Late in the lytic cycle, two distinct classes sedimenting respectively at 19 S and 16 S in a sucrose gradient have been found (1-2). Recently, Weinberg and Newbold (8) have analysed the sedimentation patterns of radioactive hybridizable SV40-specific RNA present in the cytoplasm of cells late after lytic infection. Whole SV40 DNA, its fragments obtained after treatment with the Hemophilus aegyptius restriction endonuclease or DNA from non defective Adeno-SV40 hybrid ND 1 (9) were utilized in these hybridization experiments. These authors have shown that the large viral 19 S RNA synthesized late in the lytic infection consists mainly of a species similar to the transcript of the late region of the viral genome as defined by Khoury et al. (5). This late region is represented in Hind fragments

1995

Nucleic Acids Research C, D, E, K, F, J and G (52 % of the viral genome). Again, Weinberg and

Newbold (8) have shown that the large late 19 S viral RNA shares sequences with the small late viral 16 S RNA. In this paper we have confirmed the general position of the transcription unit of the SV40-specific late 19 S mRNA and we have also mapped the SV40-specific late 16 S mRNA ; in these experiments we have purified polysomal RNA from SV40-lytically infected cells, separated 19 S and 16 S RNA classes and hybridized them with SV40 DNA and its Hind-fragments.

MATERIALS AND METHODS

SV40, large plaque, SV1 strain (10), was grown and assayed for its plaque-forming titer on CVl (monkey kidney) cell cultures. Viral. lysates were obtained by infection of CV1 cells with dilute viral preparations (0.005 PFU/Cell). [14C]-labelled viral lysates used for the preparation of Hind-fragments of SV40 DNA were obtained by l;abelling the SV40-infected CV1 cultures from the 7th to the 10th day after infection with 5 iCi of [ 14C] methyl-thymidine (55.3 mCi/mmol CEA, France) per 10-cm Petri dish. SV40 was purified in KBr gradients as already described (2). To obtain "late" SV40-specific polysomal RNA, the CVl cells were seeded in five 10-cm plastic petri dishes (Falcon) at 1.5 x 10 6 cells in minimal essential medium supplemented with 0.3 7 tryptose phosphate, 0.35 % glucose, 500 international units of penicillin/ml, 100 Vg/ml of streptomycin (Me CV1 medium) and with 10 7 calf serum (Gibco), and the cultures were placed in a CO 2 -incubator. The cultures were infected at a m.o.i. of 100-200 PFU/cell before the cells became confluent (11) at 24 hr after seeding. The virus was adsorbed to the cells at 37'C for 1 hr, the oultures were then washed and covered with Me CVl medium supplemented with 2 % calf serum. Twenty four hr after infection, the cultures were recovered with 3 ml per culture of warmed (37'C) Me CVl medium, without tryptose, -3supplemented with 2 % dialyzed calf serum containing 1500 yCi of l H3 -5uridine (CEA, France, 20 Ci/mmol). Polysomes and polysomal RNA six hr after the addition of [3H] the medium was removed and the cultures were washed twice with uridine, 5 ml/petri dish of Buffer I (10 mM triethanolamine, pH 8, 25 mM NaCl, 5 mM MgCl2, 250 mM sucrose).

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Nucleic Acids Research After the medium was removed from the cells, 1 ml/petri dish of cold Buffer I + 0.8 % NP 40 (Nonidet P40, Shell Chemical Co) + 2 mg yeast RNA (type XI, Sigma, repurified by phenol extraction) was added and left for 5 minutes. Then the lysate was carefully scraped off the surface and collected into a glass centrifuge tube (Sorval SS 34 rotor). Nuclear and cytoplasmic fractions were separated by centrifugation at 2 500 rpm for 5 minutes at 4'C. The supernatant was centrifuged at 10 000 rpm (Sorval SS 34 rotor) for 20 minutes. The resulting post mitochondrial supernatant placed into a tube containing 0.25 ml of 10 % sodium deoxycholate (DOC) (final concentration of DOC was 0.5 7). The mixture was centrifuged through a 0.5 M/2 M sucrose double layer (3 ml of 0.5 M sucrose over 2.5 ml of 2 M sucrose) in a Spinco fixed angle 65 rotor at 38 000 rpm for 2 1/2 hr at 4°C. The pellet was then suspended carefully in 1.5 ml of Buffer I.

was

(As judged from an analytical sucrose gradient centrifugation, more than of the suspension consisted of polyribosomes). 80 We added to the suspension 0.22 ml of 0.1 M EDTA, (final concentration 2 mM) 7.2 ml of 20 mM NaCl-Na acetate, pH 5, 1.8 ml of 0.15 M NaCl 40 mM) and 0.44 ml of 25 % sodium dodecyl sulfate (final concentration (SDS) (final concentration 1 7). The polysomal suspension was then extracted twice in 7.5 ml of phenol, 7.5 ml of chloroform, 0.15 ml of isoamyl alcohol (each time for 5 minutes at room temperature). The final extract was brought to 0.14 M in NaCl and then precipitated with two volumes of ethanol. The specific activity of polysomal

Sedimentation in

sucrose

33H3 RNA was gradients

about 2

x

105

cpm/ug.

Preparative sedimentation to

separate the 19 and 16 S RNA classes was performed as follows :

Polysomal RNA samples in 200 el of a buffer containing 10 mM triethanolamine, pH 7.4, 50 mM NaCl, and 1 mM EDTA (used for preparing (w/v) sucrose sucrose gradients) were centrifuged in 16 ml, 15-30 gradients in Spinco SW 27.1 rotor at 23 000 rpm for 20 hr at 200C. Fractions of 0.4 ml were collected from the top of the tubes with an ISCO 640 density gradient fractionator while absorbance at 254 nm was monitored with an UA-4 absorbance monitor. Two 5-Vl aliquots were taken from each fraction. One was hybridized with SV40 DNA as described by May et al. (2), in order to determine the sedimentation profile in a sucrose gradient of polysomal SV40 RNA. The other aliquot was assayed for total acid-insoluble radioactivity. To control the separate 19 S and 16 S SV40 mRNA preparations (see results) similar sedimentations in sucr-ose gradient were performed

.1997

Nucleic Acids Research except that the aliquots taken from each fraction were of 100 1l instead of 5 l.

Preparation of SV40 DNA i) For hybridization experiments with whole viral DNA-carrying filters, SV40 DNA was prepared as already described (2).

ii) SV40 DNA to be treated by Hind-restriction endonuclease was prepared as follows : SV40 virus was purified in a KBr gradient (2), dialyzed against 0.1 x SSC (SSC = 0.15 M NaCl, 0.015 M Na citrate), brought to 1 % SDS in a final volume of 5 ml. The solution was incubated at 450C for 30 minutes. The closed superhelical DNA molecules were then separated by equilibrium centrifugation in CsCl containing ethidium bromide as described by Cuzin et al. (12). The specific activity of the SV40 DNA obtained was about 1.6 x 104

114Cj

cpm/ig.

Degradation of

[14C]

SV40 DNA by IjLjd II + III Hind II + III restriction enzymes were purified by the method of Kopecka (13) from Hemophilus influenzae-Rd strain.

l4C]

SV40 DNA (100-150 Vg) in 6.6 mM Tris-HCl buffer, pH 7.4, 6.6 mM MgCl2, 6.6 mM 2-Mercaptoethanol and 50 mM NaCl was digested with 180-220 1l of Hind II + III in a total volume of 1 ml for 16 hr at 370C under these conditions, digestion is complete. The volume of the final digest was reduced by evaporation to about 200 l ; 20 Vl of 10 7 SDS was added and the mixture was incubated for 30 minutes at 370C ; sucrose and bromophenol blue were added to the digest to final concentrations of 7.5 % and 0.015 %, respectively. Electrophoresis was carried out in slab gels (20 x 40 x 0.4 cm) of 3 % polyacrylamide in buffer (0.04 M Tris/acetate ; 0.024 M sodium acetate,

0.002 M EDTA, pH 7.8) at 40 mA, 2000 for 24 hr. The gels were soaked in the electrophoresis buffer containing 2- g/ml of ethidium bromide (Sigma, St.Louis, Mo USA) for 5 minutes and the bands were visualized in ultraviolet light (short-wavelength lamp). Elution of bands by electrophoresis and purification of the fragments The slab gel was washed twice with 10.-3 M Tris-HCl buffer, pH 7.8 to eliminate most of the ethidium bromide. Slices corresponding to the fragments A, B, E, F, G, H, I, J, K and to the fragments C plus D were cut out from the slab under UV light. Each slice was pushed through a syringe to squash the gel and placed into special glass tubes closed at the bottom with a dialysis bag. The top of the tubes was covered with the

1998

Nucleic Acids Research electrophoresis buffer and the ends of dialysis bags were also soaked in this buffer. Electrophoresis was carried out at 130 V (10 V/tube) at 200C for 20 hr. The DNA was recovered in the dialysis bags in 5-15 ml. The radioactivity of the aliquots of each sample was counted in Bray solution. The recovery of the radioactivity varied from 42-79 % under these conditions. Each fragment was repurified by three or four cycles of adsorption to (in 0.14 M phosphate buffer, pH 6.8) and elution from a hydroxyapatite column (in 0.40 M phosphate buffer, pH 6.8) (14). Hybridization DNA-RNA For the hybridization with whole SV40 DNA, viral DNA was treated and immobilized on the 25-mm filters (Schleicher and Schuell BA 85) as already described (2). The purified fragments were immobilized on 25-mm filters after alkaline denaturation with NaOH (pH 12.5) followed by neutralization with glacial acetic acid. Square minifilters (3 x 3 mm) were cut from the dried and baked (80°C, 2 hr, under vacuum) 25-mm filters. Blank minifilters containing no DNA were treated and cut in the same way. The fragment-carrying minifilters were then preincubated in the hybridization mixture (see below) for 24 hr at 370C. After this treatment the fragments were irreversibly immobilized on the filter as judged by the cpm retained on the filters. It is of interest to note that the a smaller given fragment is, the weaker is its ability to be retained on the filter. The quantity of fragment(s) retained per minifilter was equivalent to that contained in 60-200 ng of whole SV40 DNA. Hybridization of uridine RNA with either whole SV40 DNA or its Hind-fragments was made in formamide as described by May et al. (2) with the exception that the SV40 RNA preparations were degraded before hybridization by treating them with 0.1 M NaOH at room temperature for 3 minutes and then neutralizing them with glacial acetic acid. (After this treatment the SV40 RNA species sedimented in a sucrose gradient as a broad band with a sedimentation coefficient of about 10 S). A u-inifilter carrying either the fragment(s) or the whole DNA (200 ng/filter) was incubated at 370C for 70 hr with quantities of 19 S or 16 S RNA preparation containing 54 000 - 72 000 cpm (19 S preparation) and 37 000 - 50 000 cpm (16 S preparation) in 100 Vi1 ; the filters were counted for [3H] and t 4C] . Overlap of I74C] into the [3H 1channel and of £3H] into the [14C3 channel was substracted from all results. Radioactive, hybridizable RNA is defined as the difference between the

[14C]

[3H3

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Nucleic Acids Research ribonuclease-resistant radioactivity (cpm) of the DNA-containing filter and the ribonuclease-resistant-radioactivity (cpm) of the blank filter. All assays of a givep fragment (A, B, E, F, G, H, I, J or K) or of the C + D mixture (approximately equimolar) were performed with identical fragment-carrying minifilters cut from the same 25-mm filter containing this fragment. By examining the DNA saturation curves at'various RNA concentrations and the efficiency of hybridization with different amounts of (whole) SV40 DNA on filters, we observe'd that experiments carried'out with minifilters loaded with 200 ng of whole SV40 DNA correspond to hybridizations using DNA in excess. RESULTS

The sedimentation pattern of the radioactive hybridizable RNA extracted from polysomes of CVl cells infected late (24 hr p.i.) shows essentially two classes with a sedimentation coefficient of about 19 and 16 S (fig. 1). In order to separate these classes, the fractions (a) and (b) (fig. 1) were pooled and designated as 19 S and 16 S SV40 mRNA preparations respectively. When,both preparations were resedimented in a sucrose 8radient, the radioactive hybridizable RNA sedimented as a uniform band with a sedimentation coefficient of (a) 19 S and (b) 16 S (fig. 2 'a

and b). The 19 S and 16 S SV40'-mRNA preparations contained, respectively, 10 %/ and 16 7. of radioactive RNA hybridizable with whole SV40 DNA as compared to the total acid-preci'pitable radioactivity present in these preparations. The 19 S and 16 S preparations were divided into several portions and hybridized with whole SV40 and with the Hind-fragments as described under Methods. The experimental data and calculated results are given in Table I. Each hybridization assay was carried out in triplicate. For each preparation, 19 S and 16 S, the radioactivity hybridizable to a given fragment(s) was expressed as the percentage (y) of the radioactivity hybridizable to whole SV40 DNA. To make allowance for the relative molecular weight, Y, of the fragment(s) (where Y is the percent of SV40 DNA) (15), we calculated the ratio _ cpm hybr.dizable to fragment. MW of fragment cpm

hybridizable

to whole SV40 DNA

SV40 19 S mRNA Preparation

-2000

MW of SV40 DNA Y The r values obt,ained with this

NucleicAcids Research

FRACflO

Fig. 1 : Fractionation by sucrose density gradient of SV40-infected polysomal RNA labelled late in the lytic cycle. Polysomal RNA (180 ug, 2.2 x 105 cpm/ig) in 200 1l was layered on top of a (16 ml) linear (15-30 % w/v) sucrose density gradient and centrifuged in the Spinco SW 27.1 rotor at 23 000 rpm for 20 hr at 200C. Aliquots of each fraction were assayed for total radioactivity or for radioactivity hybridizable to SV40 DNA. The fractions designated by the horizontal bars a and b were collected and considered respectively as preparations of SV40 19 S late mRNA and SV40 16 S late mRNA.

a)

II j30

z'o 20 t 10 30

20

Fraction

Fraction

Fig. 2 : Sedimentation profile of SV40 RNA in the late SV40 19 S and 16 S mRNA preparations,507M1 aliquots of these preparations were brought to 200 VI with the buffer used for preparing the gradient and layered' on top of a'(16 ml) linear (15-30 % w/v) sucrose density gradient and centrifuged in the Spinco SW 27.1 rotor at 23 000 rpm for 20 hr at 200C. Aliquots of each fraction were assayed for total radioactivity or for radioactivity hybridizable to SV40 DNA. Panel a : lat?4SV40 19 S mRNA preparation. Panel b : late SV40 16 S mRNA preparation. | C] -labeled mouse 28 S and 18 S ribosomal RNA used as marker was sedimented in a separate tube (not indicated in the figure).

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Nucleic Acids Research

TA.E I RELTIVE

PSENTTION OF THE SEQUENCES OP TME HIND-PRAENTS IN THE PREPARATIONS OF LATE 16 S AND 19 S S5V40-SPECIFIC RUA

Molar equivaM1.W. lent of

Y

atRelative %aI of SV40

ra Pragent

DNA

y (S) in the hybridization mixture cp|pto hbiial fridizab(l) ** |hybridizbl to freeamnt tp hybrSi0Dable to04 N cpu hybridizable to SV40 DNA 0 ofamn()~ 19 S

B I H A C + D E

Kt F J G

15.0

46

5.0

160

5.5 22.5 20.5 8.5 4.0 7.5 4.5

145

7.0

71

58 105 200 120 133 128

16 S

19 S

7200 + 550 7900 + 390 225 + 71+ 150 + 0 0 319 + 5400 + 420 5930 + 290 1334 + n n 726 +

n 0

(

cshbidaletfrr)1 | 100 r

DNA (ng) SV40cphyrdzbe

per filter

15 7 19 43 164

16 S 89 52 112 174 911

+ 9 + 4 + 5 4 12 + 53 40 360 + 10 381 + 73 714 + 28 716 + 75 1452 +121 594 + 44 1222 + 80 587 + 37 924 + 37

19 S 3.12 1.02 2.21 4.43 24.7 13.44

-

Y

1

16 S

19 S

16 S

1.13 0.66 1.42 2.23

0.21 0.20 0.40 0.20 1.20

0.08 0.26

0.4 0.6 0.65

0.6 0.5

0.12

6.4

1.58

0.10 0.75 0.75

7.05

12.03

1.76

3.01

1.71

13.26 11 10.87

24.47 20.59 15.57

1.77 2.44

3.26 4.57 2.22

1.84 1.88

15.35

1.55

0.5

1.43

Expressed as the equivalent quantity of vhole SV40 DNA (ng/filter) containing the actual amount of fragment-specific sequences retained on the filter. The amounts of DNA on each filter were calculated with reference to the determined specific 14C radioactivity of the SV40 DNA used either as a whole molecule or as a fragmnt. m each value represents the mean (+ SD) of three amparate hybridization assays. _ (r)16 S nd (r)a9 S are the values of r obtained for the 16 S and 19 S ERN preparations respectively.

preparation with the different fragments fall into two groups (i) one group has values ranging between 0.2 - 0.4 (fragments A, H, I, B) (ii) and the other group has values ranging between 1.2 - 2.4 (fragments C + D, E, K, F, J, G). This observation is in accordance with the notion that the 19 S SV40 mRNA preparation contains a major constituent transcribed from all or the major part of the late region of the viral genome (region represented by Hind-fragments, C, D, E, K, J, G) (5) plus 10-20 % (in radioactivity) of the early SV40 19 S mRNA transcribed from the early region of the viral genome (represented in fragments, A, H, I, B). The variation of the r values for the different fragments derived from the late region are not surprising because of the experimental errors (see standard deviations in Table I) and also because of some possible variations in the efficiency of hybridization from one fragment-carrying filter to another.

SV40 16 S mRNA preparation The r values obtained with this preparation with the different fragments fall into three group: i) one group has values between 0.08 - 0.26 (fragments A, H, I, B) ; ii) the second group has values of 0.75 (fragments C + D, E) ; iii) the third group has values between 2.22 - 3.26 (fragments K, F, J, G). Taken as a whole, these values show that this preparation contains a major constituent that derives from a region including the Hind-fragments

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Nucleic Acids Research K, F, J and G.

In order to compare the composition of the 16 S RNA preparation and 19 S RNA preparation, we calculate for each fragment the ratio =r value for 16 S preparation value for preparation ; moreover this ratio eliminates the r value for 19 S preparation possible variations of the hybridization efficiency from one filter to another. The values obtained for Q (last column, Table I) are representative of four other independent experiments. It appears that the specific sequences present in fragments K, F, J, G are represented twice as much in the

19=S r

16 S RNA preparation as in the 19 S RNA preparation. By contrast, the specific sequences present in the other fragments (late or early) are represented about half as much in the 16 S RNA preparation as in the 19 S RNA preparation. These results suggest that the SV40 16 S mRNA preparation contains a late SV40 16 S mRNA species transcribed from the region of the viral

including Hind-fragments K, F, J, G. The presence in relatively low amounts of sequences corresponding to other fragments (with similar V values) results probably from contamination by or degradation of the constituents (early and true late 19 S mRNA) present in the SV40 late 19 S mRNA preparation. However, we cannot rule out the existence of another 16 S viral late mRNA species

genome

present in relatively low amounts.

It appears likely that this region including the complete fragments F and J, and the complete or almost complete fragments K and G, is located between 0.945 and 0.175 map units. The precise location of the ends of this region cannot be determined using the present approach.

DISCUSSION Our results have been mapped as shown in Fig. 3. They are consistent with the following findings : i) Prives et al. (16) have shown that the predominant translation product of late 16 S RNA obtained a cell free extract is the VPl-protein. (ii) All mutants classified on the basis of their behaviour in complementation tests and who,se mutation is localized in the region comprising Hind-fragments F, J, G fall into B, C or BC complementation groups (17-18), and it has been shown that the complementation pattern of these mutants probably results from intracistronic complementation (17). (iii) Studies of the fingerprints of VPl-protein show that both of the late mutants groups B and C are defective in a single gene which codes for

in

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Nucleic Acids Research

ros 00. 9

2*s 0.1

Fig. 3 Mapping the transcription sites of late SV40 19 S and 16 S mRNA species.

VPI (Faye and Hirt, personnal communication). It should be noted that mutations of the other'group (D) of complementation in the late region lie on the segment corresponding to Hind-fragment E (18) adjacent to Hind-fragment K. (iv) Weinberg and Newbold (8) have shown that the SV40specific late 16 S RNA hybridized well to Adeno-SV40 hybrid ND 1 DNA which contains'SV40 sequences lying across an early-late border, between 0.11 and 0.28 map units (9). (v) The start of the VPl-gene is located within Hind K and very close to the'boundary with Hind E (Fiers, Rogiers, Soeda, Van de Voorde, Van Heuverswyn, Van Herrewe'ghe, Volckaert and Yand, personnal

communication). If we consider as a first approximation that the region of the genome corresponding to the 16 S RNA is similar to the region including complete Hind-fragments K, F, J, G from about 0.945 to 0.175 map units)

(Fig. 3), we obtain a length equivalent to 23 % of the viral genome, i.e., a molecular weight of about 400 000 Daltons for the coding portion of the

16 S late mRNA (assuming a value of 3.5 x .106 Daltons for the molecular weight of SV40 DNA). This value is consistent with the molecular weight of the VPl-protein (46 000 Daltons, ref. 16). NOTE ADDED IN PROOF Just before submitting this paper for publication we learned that Khoury et al. (19) using another approach found results similar to ours.

ACKNOWLEDGEMENTS We wish to thank Dr. R. Kamen for his helpful suggestions, Dr. L.F. Cavalieri for a critical reading of the manuscript and Miss J. Borde for skilled technical assistance.

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Nucleic Acids Research *Laboratoire de Genetique Moleculaire, Institut de Biolcgie Moleculaire, 2 Place Jussieu, 75 005-Paris, France REFERENCES 1 Weinberg, R.A., Warnaar, S.0. and Winocour, E. (1972) J. Virol., LO, 193-201.

2

May, E., May, P. and Weil, R. (1973) PrOc. Nat. Acad. Sci., USA,

70, 1654-1658. 3

Weil, R., Salomon, C., May, E. and May, P. (1974) Cold Spring Harbor Symp. Quant. Biol., 39, 381-395.

4

Khoury, G., Martin, M.A., Lee, T.N.H. and Nathans, D. (1975) Virology, 63, 263-272.

5

Khoury, G., Howley, P., Nathans, D. and Martin, M. (1975) J. Virol., 15, 433-437.

6

May, E., May, P. And Weil, R. (1975)-, in preparation.

7

The nomenclature used is that proposed-by Smith H.- .and Nathans, D. 1973, J. Mol. Biol., 81, 419-423.

8

Weinberg, R.A. and Newbold, J.E. (1974) Cold Spring Harbor Symp. Quant. Biol., 39, 161-164.-

9

Morrow, J.F., Berg, P., Kelly, T.J. and Lewis, A.M. (1973) J. Virol.,

12, 653-658. 10 Tournier, P., Cassingena, R., Wicker, R., Coppey, J. and Suarez, H. (1967) Int. J. Cancer, 2, 117-132. 11 Manteuil, S., Pages, J., Stehelin, D. and Girard, M. (1973) J. Virol., 11, 98-106.

12

Cuzin, F., Vogt, M., Dieckmann, M. and Berg, P. (1970) J. Mol. Biol.,

47, 317-333. 13 Kopecka, H. (1975) Biochim. Biophys. Acta, 391, 109-120. 14

Bernardi, G. (1971) Procedures in Nucleic Acid Research, Vol. 2, p. 455-499.

15 Danna, K.J., Sack, G. and Nathans, D. (1973) J. Mol. Biol., 78, 363-376. 16

Prives, C.L., Aviv, H., Gilboa, E., Revel, M. and Winocour, E., (1974) Cold Spring Harbor Symp. Quant. Biol., 39, 309-316.

17

Martin, R.G., Chou, J.Y., Avila, J. and Saral, R. (1974) Cold Spring Harbor Symp. Quant. Biol., 39, 17-36.

18

Lai, C.J. and Nathans, D. (1974) Cold Spring Harbor Symp. Quant. Biol., 39, 53-60.

19

Khouiry, G., Carter, B., Ferdinand, F., Howley, P. and Martin, M.A. (1975), J. Virol., in press. 2005